JP2023063189A - Crenolanib for treating flt3 mutated proliferative disorders relapsing after or refractory to prior treatment - Google Patents

Abstract

To provide methods for treating a proliferative disorder in a subject with mutated or constitutively active FLT3 who has relapsed after or is refractory to one or more prior tyrosine kinase inhibitors.SOLUTION: A method for treatment comprises: obtaining a tumor sample from the subject who has relapsed after or is refractory to one or more prior tyrosine kinase inhibitors; measuring expression of a mutated or constitutively active FLT3 mutant in the tumor sample; and administering to the subject a therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof sufficient to treat the proliferative disorder.SELECTED DRAWING: None

Description

There is an application for the application of Article 30, Paragraph 2 of the Patent Law https://doi. org/10.1182/blood-2020-139898 Published November 5, 2020

Detailed description of the invention

Cross-reference to related applications None

The present invention provides the use of crenolanib, or a salt thereof, as a single agent or in combination with another agent for the treatment of cancer and FLT3 relapsed/refractory to previous cancer therapy. A method for treating an animal suffering from a mutagenic proliferative disorder is directed.

Description of federally funded research None

Incorporation by Reference of Materials Filed on Compact Disc None

Without limiting the scope of the invention, the background art is described in relation to its ability to inhibit FLT3 tyrosine kinase in the treatment of proliferative disorders involving mutated or otherwise activated FLT3. there is

Protein kinases are enzymes that chemically modify other proteins by catalyzing the transfer of a phosphate group to the amino acid residues serine, threonine, or tyrosine. Approximately 30% of all human proteins can be modified by kinase activity, and kinase signaling is involved in several cellular processes including growth, proliferation, and survival.

Aberrant expression, or activation, of protein kinases is often the result of their involvement in cell growth and survival, including through mutation, copy number gain, amplification, gene fusion, or other mechanisms. Associated with proliferative diseases, including various cancers. Thus, the investigation of compounds that potently inhibit the activity and function of protein kinases allows for a better understanding of the physiological roles of protein kinases.

The FMS-like tyrosine kinase 3 (FLT3) gene encodes a membrane-bound receptor tyrosine kinase that affects hematopoiesis, and abnormalities in FLT3 signaling can lead to hematological disorders and malignancies (Gilliland & Griffin, 2002; Stirewalt & Radich 2003). Activation of the FLT3 receptor tyrosine kinase is initiated via binding of the FLT3 ligand (FLT3L) to the FLT3 receptor, and activation results in homodimerization, cross-phosphorylation, and down-regulation of the ligand-bound receptor. Recruitment of stream signaling factors begins.

FLT3 is one of the most frequently mutated genes in hematological malignancies, present in approximately 30% of adult acute myeloid leukemia (AML) (Papaemmanuil et al., 2016; Rucker et al., 2021). Tyner et al., 2018), the presence or absence of FLT3 mutations are included in international guidelines for AML risk stratification (Dohner et al., 2017).

The most common FLT3 mutation is an internal tandem duplication (ITD) that results in an in-frame insertion within the juxtamembrane domain of the FLT3 receptor and has been reported in 20-30% of adult AML patients. FLT3-ITD mutations are independent predictors of poor patient prognosis and are associated with increased risk of relapse and reduced disease-free time and overall survival after standard therapy (DiNardo & Lachowiez, 2019; Papaemmanuil et al., 2016; Rucker et al., 2021; Sakaguchi et al., 2019; Tyner et al., 2018). Point mutations with ligand binding or kinase domain mutations are less frequent than ITD mutations, but are also prognostically significant. The most commonly affected amino acid residue is aspartate 835 (D835) within the activation loop. Missense mutations (nucleotide substitutions) at D835 occur in approximately 5-10% of adult AML patients (Stirewalt & Radich, 2003; Tyner et al., 2018). While the ITD and D835 mutations are detectable using PCR-based techniques that allow relatively low cost and relatively rapid results, the current commercial availability of next-generation sequencing panels, and The more widespread availability of reagents and equipment required for such panels performed outside of commercial laboratories has redefined the mutational landscape of AML in general and FLT3-mutant AML in particular. A detailed genetic analysis on 500 AML patient samples found that up to 20% of the identified point mutations in FLT3 did not involve amino acid D835 (Tyner et al., 2018).

Its frequency of FLT3 activating mutations in AML makes this kinase an interesting target for drug discovery. Several FLT3 inhibitors with different degrees of potency and selectivity for activated FLT3 have and are currently being investigated in AML patients. To date, two FLT3 inhibitors have been approved by the United States Food and Drug Administration (FDA) or the European Medicines Agency (EMA) for use in FLT3-mutated AML. Approved: midostaurin is approved in combination with chemotherapy for the treatment of newly diagnosed FLT3-mutated AML; Approved for use (FDA, 2019, 2020). Midostaurin and gilteritinib are the only currently approved FLT3 inhibitors, although several other compounds have been or are currently being investigated.

Although FLT3 inhibition has proven to be a desirable therapeutic option, it has several drawbacks to single-agent targeted therapies. Most of the tested FLT3 inhibitors are tyrosine kinase inhibitors (TKIs), including midostaurin, gilteritinib, and the present invention. Tyrosine kinase inhibitors may be susceptible to resistance mutations, which are mutations in target genes that confer resistance to certain TKIs. The presence of resistance-conferring mutations not only predicts an incomplete response prior to TKI administration, but patients can acquire resistance-conferring mutations while receiving TKIs and relapse. There is also sex. In many cases, these resistance-conferring mutations cause conformational changes in the kinase that prevent TKI binding.

For example, the canonical FLT3 kinase domain mutation at amino acid residue D835 confers resistance to the TKIs quizartinib and sorafenib. Both quizartinib and sorafenib are "type II" inhibitors and bind to hydrophobic sites near the ATP binding pocket. Mutations at D835 alter the structure of this hydrophobic site, preventing inhibitor binding (C. C. Smith, Lin, Stecula, Sali, & Shah, 2015). Mutations at amino acids F691 and N701, “gatekeeper residues,” confer resistance to gilteritinib by altering the structure of the ATP-binding pocket to which gilteritinib binds (Joshi et al., 2021). Up to 12% of patients who relapse after single-agent gilteritinib therapy develop FLT3-F691 mutations that contribute to relapse (McMahon et al., 2019). Mutations at amino acid residues A637, N676, G697, Y842, and A848 are associated with resistance to several FLT3 inhibitors (Wang et al., 2021). Acquisition of secondary FLT3 mutations could explain the relatively short event-free survival (EFS) observed for single-agent FLT3 inhibitors, with a mean EFS of 2.8 months for gilteritinib monotherapy. have been reported (Perl et al., 2019).

The majority of patients treated with either midostaurin or gilteritinib remain mutated in FLT3 in relapses, either retaining the original mutated clone or acquiring secondary mutations (Altman et al. et al., 2021; McMahon et al., 2019; Schmalbrock et al., 2021). These patients would benefit from administration of FLT3 inhibitors in relapse. This is because salvage chemotherapy alone does not provide high cure rates for relapsed/refractory FLT3 mutant AML (see control arm of the gilteritinib phase III trial) (Perl et al., 2019). However, due to the potential acquisition of resistance-conferring mutations, relapsed/refractory patients to one FLT3 TKI are more susceptible to a second or even a third FLT3 TKI. It is unlikely to benefit from monotherapy (Perl et al., 2021).

A pan-FLT3 inhibitor active against some resistance-conferring FLT3 variants, alone or in combination with another agent, may be used to ensure optimal patient benefit. necessary. Thus, there remains an unmet need to treat patients with FLT3-mutated proliferative disorders who have been treated with one or more FLT3 tyrosine kinase inhibitors.

The present invention provides crenolanib (and its pharmaceutically acceptable salts), a potent pan- FLT3 inhibitor with activity against mutations conferring resistance, by prior FLT3 inhibitors. The limitations of the prior art are overcome by use in the treatment of FLT3-mutant proliferative disorders that are relapsed or refractory after treatment.

In one embodiment, the present invention provides for relapsing/refractory to one or more prior tyrosine kinase inhibitors among subjects with mutated or constitutively active FLT3. , a method of treating a proliferative disorder comprising obtaining a tumor sample from a subject relapsed/refractory to one or more prior tyrosine kinase inhibitors; mutated or constitutive in the tumor sample and administering to the subject a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, sufficient to treat the proliferative disorder. including. In one aspect, the mutated or constitutively active FLT3 is FLT3-ITD; FLT3-TKD; an activating mutation in FLT3; an increase in copy number or amplification of the FLT3 gene; At least one of the gene fusions comprising the fusion. In another aspect, the subject has midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279 , FYSYN, NMS-03592088, or TG02 citrate; or the subject has an FLT3 mutation that confers resistance to the previous tyrosine kinase inhibitor. In another aspect, the resistance-conferring FLT3 mutation is present alone or in combination with the FLT3-ITD mutation, amino acid residues K429, A627, N676, A680, F691, Y693, G697, D698, N701, D835 , N841, Y842, A848. In another aspect, the resistance-conferring FLT3 mutation was present prior to prior administration of the tyrosine kinase inhibitor, or the resistance-conferring FLT3 mutation was present during or prior to administration of the prior tyrosine kinase inhibitor. was obtained later. In another aspect, the proliferative disorder is gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer cancer, salivary gland cancer, small cell lung cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancies. In another aspect, the therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is about 50-500 mg per day, 100-450 mg per day, 200-400 mg per day, 300-500 mg per day, 350-500 mg per day, 500 mg, or 400-500 mg per day. In another aspect, the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered at least one of continuously, intermittently, systemically, or locally. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered orally, intravenously, or intraperitoneally. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered up to three times daily or more for as long as the subject is in need of treatment for a proliferative disorder. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is provided at least one of: sequentially or concurrently with another agent to maintain remission in an existing patient provided as a single agent or in combination with another agent in patients with relapsed/refractory proliferative disorders to maintain remission in patients with relapsed/refractory proliferative disorders; or relapsed/refractory proliferative disorders is provided as a single agent or in combination with another agent to maintain remission in pediatric patients with In another aspect, crenolanib or a pharmaceutically acceptable salt thereof is crenolanib besilate, crenolanib phosphate, crenolanib lactate, crenolanib hydrochloride, crenolanib citrate, crenolanib acetate, crenolanib toluene sulfonate, or klenoranib succinate.

In another embodiment, the invention provides a method of inhibiting or reducing mutant FLT3 tyrosine kinase activity or expression in a subject suffering from a proliferative disorder, comprising: identifying a subject who has discontinued prior tyrosine kinase inhibitor therapy; obtaining a tumor sample from the subject; measuring expression of mutated FLT3 or a constitutively active FLT3 mutant in the tumor sample; and if the subject has a mutated FLT3 or a constitutively active FLT3 mutant, administering to the subject a therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof, wherein crenolanib or a salt thereof includes methods of reducing proliferative disorder burden or preventing progression of proliferative diseases. In one aspect, the mutated or constitutively active FLT3 is FLT3-ITD; FLT3-TKD; an activating mutation in FLT3; an increase in copy number or amplification of the FLT3 gene; At least one of the gene fusions comprising the fusion. In another aspect, the subject has midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279 , FYSYN, NMS-03592088, or TG02 citrate; or the subject has an FLT3 mutation that confers resistance to the previous tyrosine kinase inhibitor. In another aspect, the subject is relapsed or refractory to previous tyrosine inhibitors and the subject has amino acid residues K429, A627, N676, A680 present alone or in conjunction with the FLT3-ITD mutation. , F691, Y693, G697, D698, N701, D835, N841, Y842, A848. In another aspect, the resistance-conferring FLT3 mutation was present prior to administration of the previous tyrosine kinase inhibitor, or the resistance-conferring FLT3 mutation was present during or after administration of the previous tyrosine kinase inhibitor. had been obtained. In another aspect, the proliferative disorder is gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer cancer, salivary gland cancer, small cell lung cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancies. In another aspect, the therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is about 50-500 mg per day, 100-450 mg per day, 200-400 mg per day, 300-500 mg per day, 350-500 mg per day, 500 mg, or 400-500 mg per day. In another aspect, the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered at least one of continuously, intermittently, systemically, or locally. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered orally, intravenously, or intraperitoneally. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered up to three times daily or more for as long as the subject is in need of treatment for a proliferative disorder. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is provided at least one of sequentially or concurrently with another agent to maintain remission in an existing patient. provided as a single agent or in combination with another agent in patients with relapsed/refractory proliferative disorders to maintain remission in patients with relapsed/refractory proliferative disorders; or relapsed/refractory proliferative disorders is provided as a single agent or in combination with another agent to maintain remission in pediatric patients with In another aspect, crenolanib or a pharmaceutically acceptable salt thereof is crenolanib besilate, crenolanib phosphate, crenolanib lactate, crenolanib hydrochloride, crenolanib citrate, crenolanib acetate, crenolanib toluene sulfonate, or klenoranib succinate.

In another embodiment, the invention provides a method for treating a subject suffering from a proliferative disorder, wherein the patient has a genetic mutation in the FLT3 gene, an alteration in the kinase activity of FLT3 tyrosine kinase, FLT3 tyrosine kinase or by obtaining or having obtained a biological sample from a patient to determine whether they have an overexpression of FLT3 tyrosine kinase, or a change in the phenotype or genotype of FLT3 tyrosine kinase; and performing an assay on the biological sample, or determining whether the subject has increased FLT3 tyrosine kinase activity; treating the patient with a first tyrosine kinase inhibitor; and determining if the patient is refractory after the first tyrosine kinase inhibitor and if the patient has a genetic mutation in FLT3; an alteration in the kinase activity of FLT3, an overexpression of FLT3, or an alteration in the genotypic phenotype of the FLT3 tyrosine kinase. discontinuing administration of the tyrosine kinase inhibitor of 1 and internally administering an effective amount of crenolanib to reduce the proliferative burden or prevent progression of the proliferative disease. In one aspect, the mutated or constitutively active FLT3 is FLT3-ITD; FLT3-TKD; an activating mutation in FLT3; an increase in copy number or amplification of the FLT3 gene; At least one of the gene fusions comprising the fusion. In another aspect, the subject has midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279 , FYSYN, NMS-03592088, or TG02 citrate; or the subject has an FLT3 mutation that confers resistance to the previous tyrosine kinase inhibitor. In another aspect, the mutation in the FLT3 gene or the alteration in the FLT3 phenotype or genotype is present alone or in combination with the FLT3-ITD mutation, amino acid residues K429, A627, N676, A680, F691, Y693 , G697, D698, N701, D835, N841, Y842, A848. In another aspect, the resistance-conferring FLT3 mutation was present prior to prior administration of the tyrosine kinase inhibitor, or the resistance-conferring FLT3 mutation was present during or prior to administration of the prior tyrosine kinase inhibitor. was obtained later. In another aspect, the proliferative disorder is gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer cancer, salivary gland cancer, small cell lung cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancies. In another aspect, the therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is about 50-500 mg per day, 100-450 mg per day, 200-400 mg per day, 300-500 mg per day, 350-500 mg per day, 500 mg, or 400-500 mg per day. In another aspect, the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered at least one of continuously, intermittently, systemically, or locally. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered orally, intravenously, or intraperitoneally. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered up to three times daily or more for as long as the subject is in need of treatment for a proliferative disorder. In another aspect, a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is provided at least one sequentially or concurrently with another agent to maintain remission in an existing patient; Provided as a single agent or in combination with another agent in patients to maintain remission in patients with relapsed/refractory proliferative disorders; or in pediatric patients with relapsed/refractory proliferative disorders It is provided as a single agent or in combination with another agent to maintain remission. In another aspect, crenolanib or a pharmaceutically acceptable salt thereof is crenolanib besilate, crenolanib phosphate, crenolanib lactate, crenolanib hydrochloride, crenolanib citrate, crenolanib acetate, crenolanib toluene sulfonate, or klenoranib succinate.

In another embodiment, the invention provides a method for treating a subject suffering from leukemia, comprising obtaining a sample from the subject; determining that the subject has a certain FLT3 receptor; further determining that the subject is refractory to administration or has relapsed after administration of a previous tyrosine kinase inhibitor; administering to the subject a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, sufficient to treat the leukemia. In one aspect, the mutated or constitutively active FLT3 is FLT3-ITD; FLT3-TKD; an activating mutation in FLT3; an increase in copy number or amplification of the FLT3 gene; At least one of the gene fusions comprising the fusion. In another aspect, the subject has midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279 , FYSYN, NMS-03592088, or TG02 citrate; or the subject has an FLT3 mutation that confers resistance to the previous tyrosine kinase inhibitor. In another aspect, the mutation in the FLT3 gene or the alteration in the FLT3 phenotype or genotype, alone or in combination with the FLT3-ITD mutation, is present at amino acid residues K429, A627, N676, A680, F691, Y693, A resistance-conferring mutation selected from missense mutations occurring in at least one of G697, D698, N701, D835, N841, Y842, A848. In another aspect, the resistance-conferring FLT3 mutation was present prior to prior administration of the tyrosine kinase inhibitor, or the resistance-conferring FLT3 mutation was present during or prior to administration of the prior tyrosine kinase inhibitor. was obtained later. In another aspect, the leukemia is Hodgkin's disease, myeloma, acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic neutrophilic leukemia (CNL) acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL), prolymphocytic leukemia (PML); juvenile myelomonocytic leukemia (JMML); adult T-cell ALL, acute myeloid leukemia (AML) , AML with trilineage myelodysplasia, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), or multiple myeloma (MM).

While the making and use of various embodiments of the present invention are discussed in detail below, the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. be recognized to provide The specific embodiments discussed in this specification are merely exemplary of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate understanding of the invention, some terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. For example, terms such as "a," "an," and "the" are not intended to refer only to singular entities, but to include general classes for which specific examples may be used for purposes of illustration. Intend. While these terms are used herein to describe specific embodiments of the invention, their use defines the scope of the invention, except as outlined in the claims. not a thing

The present invention provides crenolanib, or a pharmaceutically acceptable salt thereof, for treating cancer, preventing recurrence of cancer, and/or preventing exacerbation of cancer. It is intended to be administered to a subject.

Crenolanib is an orally bioavailable TKI that targets FLT3. Crenolanib is significantly more selective for FLT3 than other kinases, including c-KIT, VEGFR2, TIE2, FGFR2, EGFR, erbB2, and SRC (Lewis et al., 2009). As a type I TKI, crenolanib binds to both active and inactive conformations of kinases. Importantly, crenolanib shows preclinical activity against quizartinib- and gilteritinib-resistant FLT3 mutations, including missense mutations at D835 or F691 (C.C. Smith et al., 2014). In a direct enzyme inhibition assay, crenolanib was found to inhibit >99% of the kinase activity of the FLT3-F691L mutant at a concentration of 10 nM. Overexpress FLT3-F691L in a cell line. Crenolanib blocks FLT3 phosphorylation at nanomolar concentrations. Thus, crenolanib ideally would induce a constitutively active FLT3 proliferative disorder due to disease progression as a result of secondary resistance-conferring mutations that discontinued treatment with other FLT3 TKIs. Suitable for treatment of afflicted subjects. As a pan-FLT3 inhibitor, crenolanib has shown activity in subjects with cancers associated with FLT3 copy number gains, amplifications, fusions, or constitutively active mutants.

The present invention includes methods for inhibiting mutant or constitutively active FLT3 in a cell or subject, or methods for treating disorders associated with FLT3 activity or expression in a subject. In one embodiment, the invention provides a method for reducing or inhibiting kinase activity of mutant FLT3 in a subject, comprising administering to the subject a compound of the invention. In other embodiments, the invention provides therapeutic methods for treating a subject having a proliferative disorder driven by aberrant kinase activity of mutant FLT3. The present invention also provides methods for treating patients suffering from proliferative disorders that are relapsed/refractory to prior tyrosine kinase inhibitors.

As used herein, the term "subject" refers to animals, eg, mammals or humans, which are objects of treatment, observation or experimentation.

As used herein, the term "contacting" refers to adding crenolanib or a pharmaceutically acceptable salt thereof to cells such that the compound is taken up by the cells.

As used herein, the term "therapeutically effective amount" refers to an amount of crenolanib or a pharmaceutically acceptable salt thereof, which amount is recommended by a researcher, veterinarian, medical doctor or other clinician. is an amount that elicits a biological or medicinal response in a subject, which response is a reduction in the symptoms of the disease or disorder being treated, a reduction in the burden of a proliferative disorder (e.g., tumor size), and/or increased progression-free survival or overall survival, including long-term stable disease. Methods are known in the art for determining therapeutically effective doses for pharmaceutical compositions containing the compounds of the invention.

As used herein, the term "composition" means a product comprising the specified ingredients in the specified amounts, as well as the combination of the specified ingredients in the specified amounts, either directly or indirectly. is intended to encompass any product produced in

As used herein, the term "mutant FLT3-associated disorder" or "mutant FLT3-driven cell proliferative disorder" refers to mutant FLT3 activity, e.g., constitutive activity of FLT3 including diseases associated with or related to mutations that lead to mutations.

As used herein, the term "cell proliferative disorder" refers to one or more of cells within a multicellular organism that cause harm (i.e., discomfort or reduced life expectancy) to the multicellular organism. Refers to excessive cell proliferation of a subset. Cell proliferative disorders can occur in different types of animals and humans. Examples of cytoproliferative disorders include gastrointestinal stromal tumor (GIST), leukemia, myeloma, myeloproliferative disorders, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, Cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, salivary gland cancer, small cell lung cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancies.

As used herein, the term “relapsing/refractory” or “recurring” means that a drug was previously administered to treat a proliferative disorder but did not respond to treatment (unresponsive Responsive), or progressed after an initial response (relapse).

Detection of mutant FLT3 can be performed using any suitable means known in the art. For example, detection of genetic mutations is accomplished by detecting nucleic acid molecules (eg, DNA) using nucleic acid amplification methods (eg, RT-PCR) or high-throughput sequencing (ie, "next-generation sequencing"). can do. As an example, next-generation sequencing platforms such as Illumina can be used to determine the precise genetic sequence of a particular gene, or portion of a gene of interest. Briefly, DNA from tumor samples is fragmented, ligated with appropriate primers and adapters, and amplified using PCR during "library preparation." The prepared libraries are then sequenced using one of several commercially available systems that generate sequences for selected target genes, entire exomes, or entire genomes. The sequences are then analyzed using commercially available software, aligning the tumor sample sequences to the known sequences of the gene of interest, performing a variant calling step, and determining the DNA level in the tumor samples. to identify differences and determine whether such mutations result in altered amino acid sequences in the translated protein. Using these systems, one skilled in the art can determine whether a subject has one of the identified mutations in FLT3. Further information about FLT3, including full gene and protein sequences, known clinically relevant variants and mutations, tissue expression, and signaling interaction partners can be found at UniProt (Accession No. P36888-1), GenBank (Accession No. NM_04119.2), and GenPept Accession No. rNP_004110.2).

As used herein, the term "missense mutation" refers to an alteration in the gene sequence of the FLT3 gene that results in the substitution of one amino acid with a different amino acid when the sequence is converted into a protein. Point.

As used herein, the term "missense mutation" refers to an alteration in the gene sequence of the FLT3 gene that results in the substitution of one amino acid with a different amino acid when the sequence is converted into a protein. Point.

As used herein, the term "ITD" or "internal tandem duplication" refers to a nucleotide insertion at the DNA level in which the number of nucleotides is a multiple of three, which is equivalent to an amino acid at the protein level. addition, but does not shift the open reading frame of the gene.

As used herein, the term "resistance mutation" or "mutation-conferring resistance" or "secondary mutation" refers to a mutation susceptible to gilteritinib, midostaurin, quizartinib or other TKIs other than the invention. No, it refers to non-ITD mutations in the FLT3 gene. In other words, these mutations, whether present alone or in combination with ITD, retain kinase activity when treated with midostaurin, gilteritinib, or other TKIs, but are inhibited by the present invention. be done. Non-limiting examples of resistance mutations are missense mutations at amino acid residues K429, A627, N676, A680, F691, Y693, G697, D698, N701, D835, N841, Y842, or A848. Additional mutations in immunoglobulin-like domains, juxtamembrane domains, tyrosine kinase domains, and hinge regions are also included within the scope of the invention.

As used herein, the terms "copy number increase" or "copy number variant" refer to the presence of more than 2 copies but less than 5 copies of the FLT3 gene. As used herein, "amplification" refers to increasing the FLT3 gene or signal to more than 5 copies per cell. The increase and/or amplification of numbers can be achieved by any means known in the art, e.g., by incubating fluorescently labeled probes that bind to specific regions of DNA area) can be detected via fluorescence in situ hybridization (FISH) counting.

FLT3 kinase inhibitors known in the art are restortinib (also known as CEP-701, Kyowa Hakko, licensed to Cephalon); CHIR-258 (Chiron Corp.); Systems Inc.); midostaurin (also known as PKC412, Novartis AG); tanzutinib (also known as MLN-518, licensed to COR Therapeutics Inc., Millennium Pharmaceuticals Inc.); fizer USA XL-999 (Exelixis USA); GTP 14564 (Merck Biosciences UK); AG1295 and AG1296; CEP-5214 and CEP-7055 (Cephalon); (also known as ASP2215 FF-10101-01 (Fujifilm Pharmaceuticals); HM43239 (Hanni Pharmaceuticals); Pacritinib (also known as SB1518, CTI Biopharma); MAX-40279 (Max inovel Pty. Ltd.); LT-171-861; and TG02 citrate (Tragara Pharmaceuticals). Also (Griswold et al., 2004; Levis et al., 2002; Levis & Small, 2004; Majothi et al., 2020; Murata et al., 2003; O'Farrell et al., 2003; B.D. Smith et al. , 2004; Stone et al., 2005; Yee et al., 2002).

The inhibitors described above have been or are currently being investigated in preclinical settings or as monotherapy for recurrent AML in clinical phases I and II or in Phase III combination trials for recurrent AML. Although successful inhibition of FLT3 by these compounds has been reported in preclinical experiments, few FLT3 mutant AML patients achieve complete remission in the clinical setting. Clinical response is short-lived for most patients. Response criteria for AML clinical trials are adopted from the International Working Group on AML (Cheson et al., 2003). Responders are patients who achieve complete response (CR), complete response with incomplete blood count recovery (CRi), or partial remission (PR). Briefly, the criteria are as follows:
1. Complete Remission (CR):
a. Peripheral blood count:
i. No circulating blasts ii. Neutrophil count ≧1.0×10 ⁹ /L
iii. Platelet count ≧100×10 ⁹ /L
b. Bone marrow aspiration and biopsy:
i. < 5% blasts ii. No Auer's bodies iii. No extramedullary leukemia2. Complete remission with incomplete blood count recovery (CRi):
a. Peripheral blood count:
i. No circulating blasts ii. Neutrophil count <1.0×10 ⁹ /L, or iii. Platelet count <100 x 10 ⁹ /L
b. Bone marrow aspiration and biopsy i. < 5% blasts ii. No Auer's bodies iii. 3. No extramedullary leukemia. Partial remission:
a. All CR criteria, if abnormal before treatment, except:
b. Myeloblasts were reduced >50% but still >5%.

To date, the clinical response of FLT3 inhibitors has been largely limited to the clearance of peripheral blood (PB) blasts, which often return within weeks, while bone marrow (BM) blasts are largely unaffected. remain. For example, treatment with sorafenib, a previously described multi-kinase inhibitor with activity against mutant FLT3, was effective in eliminating PB blasts, but moderately reduced BM blasts. effect (Borthakur et al., 2011). BM blast percentage plays a central role in the diagnosis and classification of AML. The presence of an increasing percentage of blasts in the BM is associated with significantly shorter overall survival (Amin et al., 2005; Small, 2006). To effectively treat patients with FLT3-mutant AML and overcome a significant unmet need in this patient population, both PB and BM blasts should be significantly deprived of the high-risk, previously significant There is a need for inhibitors that can help bridge treated patients to stem cell transplantation, reduce relapse rates, and increase overall survival in patients with early stage disease.

As used herein, the terms "proliferative burden" or "proliferative disease burden" refer to the overall impact on the health of a subject or patient with cancer. Affecting the health of a subject or patient may include, for example, decreased overall survival, years of disability due to disease, to name a few, when compared to subjects without a proliferative disorder or disease. may include an increase in health, a decrease in wellness or overall health.

In one embodiment, the present invention provides compounds having Formula I:

or a therapeutically effective amount of a pharmaceutically acceptable salt or solvate thereof for gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES) , bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreas cancer, prostate cancer, kidney cancer, salivary gland cancer, small cell lung cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and at least one hematological malignancy is a therapeutically effective amount for proliferative diseases. Pharmaceutically acceptable salts such as hydrochlorides, phosphates and lactates are prepared in an analogous manner to benzenesulfonates and are well known to those of ordinary skill in the art. Pharmaceutically acceptable salts such as hydrochlorides, phosphates and lactates are prepared in an analogous manner to benzenesulfonates and are well known to those of ordinary skill in the art. The following representative compounds of the invention are klenoranib besilate, klenoranib phosphate, klenoranib lactate, klenoranib hydrochloride, klenoranib citrate, klenoranib acetic acid, klenoranib toluenesulfonate and klenoranib The inclusion of crenolanib as a nib succinate is for illustrative purposes only and is in no way intended to limit the invention.

The compounds of the invention can be administered to a subject systemically, eg, orally, intravenously, subcutaneously, intramuscularly, intradermally or parenterally. The compounds of the invention can also be administered topically to a subject.

The compounds of the invention can be formulated as sustained or immediate release, intended to maintain the compounds of the invention in contact with the targeted tissue for the desired range of time.

Compositions suitable for oral administration include solid forms such as pills, tablets, caplets, capsules, granules and powders, liquid forms such as solutions, emulsions and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions and suspensions.

The daily dosage of the compounds of this invention may vary over a wide range from 50-500 mg per adult human per day. For oral administration, the composition is preferably provided in the form of tablets containing 20 and 100 milligrams. The compounds of this invention may be administered on a regimen of up to three times per day or more. Preferably, it may be administered three times per day. The optimal dose to be administered may be determined by one skilled in the art and may vary with the compound of the invention used, mode of administration, time of administration, strength of preparation, particulars of the condition. Factors related to patient characteristics, such as age, weight, and diet, require dose adjustment. In other examples, the daily dosage of a compound of the invention is 15-500, 25-450, 50-400, 100-350, 150-300, 200-250, 15, 25, 50, 75, It can vary over a wide range of 100, 150, 200, 250, 300, 400, 450 or 500 mg. The compounds of the invention can also be administered on an every other day regimen once, twice, three times or more times per day. The optimal dosage to be administered can be determined by one skilled in the art and may vary with the compound of the invention used, mode of administration, time of administration, strength of preparation, particulars of the condition. One or more factors related to subject characteristics, such as age, weight, and diet, require dose adjustment. Techniques and compositions for making useful dosage forms using crenolanib are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remingtons Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); relevant portions are incorporated herein by reference.

A dosage unit for the use of crenolanib may be a single compound or a mixture thereof with other compounds, such as potentiators. Compounds can also be mixed together to form ionic or covalent bonds. The compounds of this invention can be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. Using different dosage forms, such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions, depending on the particular location or method of delivery, A patient in need of therapy can be provided with crenolanib of the present invention.

Crenolanib is generally formulated with suitable pharmaceutical salts, buffers, diluents, fillers, excipients and/or carriers (described herein) based on the intended form of administration and selected to be consistent with conventional pharmaceutical practice. are administered in admixture with a pharmaceutically acceptable carrier or carrier material). Depending on where it is best administered, crenolanib can be formulated to provide maximum and/or consistent dosing for a particular form, e.g., oral, rectal, topical, intravenous injection or parenteral administration. can be Crenolanib can be administered alone, but can generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier can be either solid or liquid, depending on the type and/or place of administration selected.

Preparation of compounds of the invention. General synthetic methods that can be referred to for the preparation of compounds of Formula I are US Pat. (published April 8, 1999) (Merck & Co.) WO 01/40217 (published July 7, 2001) (Pfizer, Inc.), U.S. Patent Application No. US2005/0124599 (Pfizer, Inc.) and U.S. patents No. 7,183,414 (Pfizer, Inc.), relevant portions of which are incorporated herein by reference.

Pharmaceutically acceptable salts such as hydrochlorides, phosphates and lactates are prepared in an analogous manner to benzenesulfonates and are well known to those of ordinary skill in the art. The following representative compounds of the invention are for illustrative purposes only and are in no way intended to limit the invention.

Example overview

Example A: Patients with FLT3-ITD and FLT3F691 mutations. After disease progression during treatment with two previous FLT3 tyrosine kinase inhibitors, midostaurin and gilteritinib, and cytotoxic chemotherapy, patients received blood, bone marrow, and central Complete clearance of leukemic blasts in the nervous system (CNS) was achieved.

Example B: A patient with an FLT3-ITD mutation that persisted after disease progression during treatment with previous FLT3 tyrosine kinase inhibitors, gilteritinib, and cytotoxic chemotherapy. The patient achieved complete remission with full count recovery after crenolanib besilate combination therapy.

Example C: A patient with an FLT3-ITD mutation that persisted after disease progression during treatment with two previous FLT3 tyrosine kinase inhibitors, midostaurin and gilteritinib, and cytotoxic chemotherapy. The patient achieved complete remission and was bridged to hematopoietic stem cell transplantation after crenolanib besilate combination therapy.

Example D: Patient with FLT3-ITD Mutation. After disease progression during treatment with three previous FLT3 tyrosine kinase inhibitors, sorafenib, gilteritinib and midostaurin, and cytotoxic chemotherapy, the patient had incomplete count recovery after crenolanib besilate combination therapy. achieved complete remission with

Example E: Patients with FLT3-ITD, FLT3D835, and FLT3Y842 mutations. After disease progression during previous treatment with FLT3 tyrosine kinase inhibitors, sorafenib, and cytotoxic chemotherapy, the patient achieved partial remission following crenolanib besylate monotherapy.

Example F: Patients with FLT3-ITD, FLT3D835, and FLT3N841 mutations. After disease progression during treatment with previous FLT3 tyrosine kinase inhibitors, sorafenib, and cytotoxic chemotherapy, the patient had complete remission with incomplete hematologic recovery after crenolanib besylate monotherapy. Achieved.

Example A: Effect of Clenoranib Besilate Therapy After Prior Midostaurin and Gilteritinib Administration in Relapsed/Refractory Patients With FLT3 Mutations Conferring Acquired Resistance: Blood, Bone Marrow, and CNS Leukemia achievement of blast clearance.

A 54 year old female was diagnosed with recurrent AML positive for FLT3-ITD and FLT3-F691 missense mutations, specifically F691L. This mutation, sometimes called a “gatekeeper” mutation, is known to confer resistance to the FLT3 tyrosine kinase inhibitor gilteritinib, among others (McMahon et al., 2019).

In March 2019, the patient was initially diagnosed with FLT3-ITD positive AML and treated with a standard chemotherapy regimen plus the FLT3 inhibitor midostaurin and achieved complete remission after 2 cycles. The patient relapsed approximately 6 months later in October 2019, at which time the patient remained FLT3 positive and received a salvage combination regimen that included the FLT3 inhibitor gilteritinib. The patient achieved partial remission and was stable on this regimen, but experienced disease progression in May 2020, at which time the patient was enrolled in a Phase 1 trial of a menin inhibitor. One month after the study, the patient experienced significant disease progression, including CNS involvement, and was removed from the study.

As of June 2020, the patient had been on three previous lines of therapy, including two FLT3 inhibitors. Molecular studies revealed that after gilteritinib treatment, the patient acquired a secondary resistance-conferring FLT3 mutation, F691L. The persistence of the FLT3-ITD mutation, the acquisition of the F691L mutation, and the fact that the patient is relapsed/refractory to multiple FLT3 inhibitors make this patient less likely to respond to treatment, and It was placed in a particularly high risk group with decreased overall survival. (Perl et al., 2021)

Because there were no other approved standard treatment options available, the treating physician applied for Special Use Authorization for crenolanib besilate, which was granted in June 2020. The patient was treated with salvage chemotherapy consisting of high-dose cytarabine and crenolanib besilate, 80 mg three times daily. At the start of treatment, patients had 72% myeloblasts, 23% peripheral blasts, and 88% blasts (of all nucleated cells) in the cerebrospinal fluid (CSF). , indicated a significant CNS involvement of these leukemias.

Bone marrow, peripheral blood, and CSF samples taken on day 21 of treatment revealed complete clearance of leukemic blasts from all three compartments. Molecular studies revealed clearance of all FLT3 clones in bone marrow. A second bone marrow biopsy taken on day 43 of treatment showed that the patient remained morphologic leukemia-free (complete remission without count recovery) and CNS leukemia-free. was confirmed. The patient was treated for 90 days and died of sepsis in remission 3.5 months after starting crenolanib therapy.

Table A below illustrates the ability of crenolanib to clear malignant leukemic blasts from the bone marrow, peripheral blood, and CSF of patients with FLT3 mutations that confer resistance after prior treatment with a tyrosine kinase inhibitor. there is

Example B: Effects of crenolanib besilate therapy in relapsed/refractory patients who were refractory to gilteritinib treatment: achieving complete remission with recovery of total counts.

A 35-year-old female was diagnosed with recurrent AML-positive for the FLT3-ITD mutation. In March 2018, the patient was initially diagnosed with FLT3-ITD positive AML, treated with standard chemotherapy regimens and achieved complete remission. Approximately three months later, in June 2018, the patient relapsed and received the FLT3 inhibitor gilteritinib as salvage therapy. The patient was refractory to this treatment, with persistent 20% myeloblasts after 2 cycles of gilteritinib therapy. Patients also experienced pericarditis as a side effect to gilteritinib treatment. After discontinuation of gilteritinib, the patient received standard salvage chemotherapy, achieved second remission, and underwent hematopoietic stem cell transplantation in November 2018. Approximately 17 months later, the patient relapsed with FLT3-ITD positive disease.

At this time in April 2020, the patient had been on three previous lines of therapy, including the FLT3 inhibitor gilteritinib. Molecular studies revealed that the patient's original FLT3-ITD mutation persisted. The persistence of this mutation and the fact that the patient relapsed after HSCT placed this patient in a particularly high-risk group with reduced likelihood of responding to treatment and shortened overall survival (Bejanyan et al. al., 2015).

Due to the scarcity of treatment options available, the treating physician applied for Special Use Authorization for crenolanib besilate, which was granted in April 2020. Patients were treated with salvage chemotherapy consisting of fludarabine, cytarabine, idarubicin, and granulocyte colony-stimulating factor, followed by crenolanib besilate, 100 mg three times daily. At the start of treatment, the patient had 75% myeloblasts, 85% peripheral blasts, and diagnostic imaging showed extramedullary disease in the spleen and lymph nodes (leukemic blasts outside the bone marrow or blood). .

A bone marrow biopsy obtained on day 36 of treatment revealed complete clearance of peripheral blasts, clearance of less than 5% of myeloblasts, and recovery of neutrophils and platelets, which was higher than the total counts. It was classified as complete remission with recovery. Molecular studies revealed clearance of all FLT3 clones. A second bone marrow biopsy obtained on day 81 of treatment confirmed that the patient remained in complete remission and had complete clearance of all extramedullary disease. The patient remained in remission for 4 months during treatment with crenolanib besilate therapy.

Table B below illustrates the ability of crenolanib to clear malignant leukemia blasts from the bone marrow and peripheral blood of patients with relapsed/refractory disease after prior tyrosine kinase inhibitor therapy.

Example C: Effect of Crenolanib Besilate Therapy in Relapsed/Refractory Patients with FLT3-ITD Mutations After Prior Gilteritinib Administration: Achieving Complete Remission with Recovery of Total Counts, and A bridge to transplantation.

A 22-year-old female was diagnosed with recurrent AML-positive for FLT3-ITD mutation. In November 2019, the patient was initially diagnosed with FLT3-ITD mutated AML and treated with a standard chemotherapy regimen containing cytarabine and daunorubicin plus the FLT3 inhibitor midostaurin and achieved complete remission after 2 cycles. However, the patient remained MRD (measurable residual disease) and FLT3 positive. The patient then received high-dose cytarabine intensive therapy in combination with midostaurin, but the MRD and FLT3-ITD mutations persisted. In May 2020, as FLT3-ITD mutations were still detectable in bone marrow samples obtained from patients, to eliminate residual FLT3-ITD positive blasts as they could potentially cause relapse. Patients were administered the FLT3 inhibitor gilteritinib with the aim of A bone marrow biopsy performed 4 weeks after single-agent gilteritinib therapy revealed that the patient had relapsed disease with 40% myeloblasts, and gilteritinib was discontinued.

At this time in June 2020, the patient had been on two previous lines of therapy, including two FLT3 inhibitors. Molecular studies showed that FLT3-ITD mutations present at diagnosis persisted through all lines of therapy (more detailed sequencing, including testing to reveal the presence of resistance-conferring point mutations, was performed). did not) was confirmed. The persistence of the FLT3-ITD mutation and the fact that the patient is relapsed/refractory to multiple FLT3 inhibitors has led to this patient's placed in a particularly high-risk group (Perl et al., 2021).

Because there were no other approved standard treatment options available, the treating physician applied for Special Use Authorization for crenolanib besilate, which was granted in June 2020. Patients were treated with salvage chemotherapy consisting of fludarabine, cytarabine, idarubicin, and granulocyte colony stimulating factor, followed by crenolanib besilate, 100 mg three times daily. At the start of treatment, the patient had 43% myeloblasts.

A bone marrow biopsy sample obtained on day 56 of treatment showed that the myeloblast percentage had fallen below 5%, and the patient underwent hematopoietic stem cell transplantation. The patient then received single-agent crenolanib besilate therapy as post-transplant maintenance starting on day 49 post-transplant with the aim of preventing another relapse. A bone marrow biopsy performed 30 days post-transplant prior to initiation of crenolanib maintenance therapy found the patient to remain in remission, although the FLT3-ITD mutation was still detectable. A second bone marrow biopsy performed 68 days post-transplant, 19 days after initiation of crenolanib maintenance, revealed elimination of the FLT3-ITD mutation.

Table C below demonstrates the ability of crenolanib to clear malignant leukemic blasts from the bone marrow of patients who had relapsed/refractory to two prior tyrosine kinase inhibitors and FLT3-ITD after hematopoietic stem cell transplantation. Figure 1 illustrates the ability of crenolanib to eliminate MRD.

Example D: Effect of Clenoranib Besilate Therapy in Relapsed/Refractory Patients with FLT3-ITD Mutations After Previous Sorafenib, Gilteritinib, and Midostaurin Administration: With Incomplete Count Recovery Achievement of complete remission.

A 76-year-old male was diagnosed with recurrent AML-positive for the FLT3-ITD mutation. In 2008, the patient was initially diagnosed with myelodysplastic syndrome, which in August 2015 converted to FLT3-ITD mutated AML. At the time of conversion to AML, the patient had been treated with standard chemotherapy regimens, achieved complete remission, and progressed to hematopoietic stem cell transplantation. Approximately nine months after transplantation, in August 2016, the patient relapsed due to CNS involvement of her leukemia and was treated with cytarabine, methotrexate, and the FLT3 inhibitor sorafenib, and achieved remission again. Two and a half years later, in April 2019, the patient relapsed, again with CNS involvement. Patients were treated with venetoclax, donor lymphocyte infusion (a common remedy for patients who relapse after hematopoietic stem cell transplantation, infusing the recipient with white blood cells from the original transplant donor), and the FLT3 inhibitor gilteritinib; Complete remission was achieved again. Seven months later, in November 2019, the patient relapsed again with CNS involvement, received cytarabine, cladribine, and a third FLT3 inhibitor, midostaurin, and again achieved a CR. Eight months later, in July 2020, the patient had a fourth relapse with 16% myeloblasts and the original FLT3-ITD mutation persisted.

As of July 2020, the patient had received four previous lines of therapy, including three FLT3 inhibitors. Molecular studies confirmed that the FLT3-ITD mutation present at diagnosis persisted through all lines of therapy (including studies revealing the presence of resistance-conferring point mutations, see more details). no sequencing was performed). The persistence of the FLT3-ITD mutation and the fact that the patient is relapsed/refractory to multiple FLT3 inhibitors has led to this patient's Placed in particularly high risk group (Perl et al., 2021)

Because there were no other approved standard treatment options available, the treating physician applied for Special Use Authorization for crenolanib besilate, which was granted in July 2020. Patients were treated with salvage chemotherapy consisting of fludarabine, cytarabine, idarubicin, and granulocyte colony-stimulating factor, followed by crenolanib besilate, 100 mg three times daily. At the start of treatment, the patient had 16% myeloblasts.

A bone marrow biopsy obtained on day 21 of treatment revealed a clearance of less than 5% of myeloblasts, along with recovery of neutrophil counts, which is considered complete remission with incomplete hematologic recovery. Classified. At this time point, the FLT3-ITD mutation was also eliminated. Due to the patient's advanced age, a routine bone marrow biopsy was not obtained and the patient remained on crenolanib treatment for approximately 6 months.

Patient Example D illustrates the ability of crenolanib to clear leukemic blasts from the bone marrow of a patient relapsed/refractory to three prior FLT3 tyrosine kinase inhibitors.

Example E: Effect of crenolanib besilate monotherapy in relapsed/refractory patients with FLT3 mutations conferring acquired resistance after prior sorafenib administration: achieving partial remission.

An 87 year old female was diagnosed with recurrent AML positive for FLT3-ITD and FLT3-D835 and Y842 missense mutations, specifically D835Y and Y842C. These mutations are known to confer resistance to the FLT3 tyrosine kinase inhibitors sorafenib and quizartinib, among others (Wang et al., 2021).

In August 2013, the patient was first diagnosed with FLT3-ITD AML, treated with a standard chemotherapy regimen, and achieved complete remission after 2 cycles. Patients were given sorafenib as maintenance therapy with the aim of preventing recurrence. Five months later, in March 2014, the patient relapsed.

At this time, molecular studies revealed persistence of the original FLT3-ITD mutation and acquisition of secondary resistance-conferring FLT3 mutations D835Y and Y842C after treatment with sorafenib. Persistence of the FLT3-ITD mutation, acquisition of the D835Y and Y842C mutations, and the fact that the patient relapsed to previous FLT3 inhibitors, reduced the likelihood of responding to treatment and reduced overall survival. were included in the particularly high-risk group with shortened s (Perl et al., 2021).

Because there were no other approved standard treatment options available, the patient was enrolled in a trial of crenolanib besilate monotherapy 100 mg three times daily (NCT01657682). At study entry, the patient had 68% myeloblasts and 30% peripheral blasts.

A bone marrow biopsy obtained on day 27 of treatment revealed a reduction in the patient's myeloblasts to 7% and elimination of peripheral blasts, which was classified as partial remission. Unfortunately, the patient died of leukemia-related complications on day 61 of treatment before obtaining a further bone marrow biopsy.

Patient Example E showed a significant reduction in malignant leukemia blasts from 30% to 7% in the bone marrow of a patient with a dual resistance-conferring FLT3 mutation after prior tyrosine kinase inhibitor therapy, and peripheral It illustrates the ability of crenolanib to completely eliminate malignant leukemia blasts in the blood.

Example F: Effect of Crenolanib Besilate Monotherapy in Relapsed/Refractory Patients with FLT3 Mutations Conferring Acquired Resistance After Prior Sorafenib Administration: Complete with Incomplete Blood System Recovery Achievement of remission.

A 31-year-old male was diagnosed with AML-positive relapsing/refractory to FLT3-ITD, and FLT3-D835 and N841 missense mutations, specifically D835V, D835Y, D835H, and N841K. These mutations are known to confer resistance to the FLT3 tyrosine kinase inhibitors sorafenib and quizartinib, among others (Wang et al., 2021).

In November 2012, the patient was first diagnosed with FLT3-ITD AML, treated with standard chemotherapy regimens, achieved complete remission, and proceeded to hematopoietic stem cell transplantation. Six months after transplantation, in November 2013, the patient relapsed and was treated with sorafenib and decitabine as salvage therapy but did not respond to treatment after multiple cycles.

In May 2014, at this time, molecular studies revealed persistence of the original FLT3-ITD mutation as well as acquisition of multiple secondary resistance-conferring FLT3 mutations following sorafenib treatment: D835V, D835Y, D835H, and N841K. The persistence of the FLT3-ITD mutation, the acquisition of the D835 and N841 mutations, and the fact that the patient relapsed to previous FLT3 inhibitors, render this patient less likely to respond to therapy and less likely to have overall survival. It was placed in the particularly high risk group with shortened duration. (Perl et al., 2021)

Because there were no other approved standard treatment options available, the patient was enrolled in a trial of crenolanib besilate monotherapy with 100 mg tid (NCT01657682). At study entry, the patient had 84% myeloblasts and 96% peripheral blasts.

A bone marrow biopsy obtained on day 29 of treatment revealed that the patient's myeloblasts had fallen to 23%, with clearance of the peripheral blasts, classified as partial remission. A second bone marrow biopsy obtained on day 57 of treatment revealed that the patient's myeloblasts had dropped to 7%, still classified as partial remission. A third bone marrow biopsy, obtained on day 84 of treatment, revealed that the patient's myeloblasts had fallen to less than 5%, along with neutrophil recovery, indicating a complete failure with incomplete hematologic recovery. Classified as remission.

Table F below illustrates the ability of crenolanib to clear malignant leukemic blasts from the bone marrow of patients with FLT3 mutations conferring resistance after prior tyrosine kinase inhibitor therapy.

It is envisioned that any embodiment discussed herein can be implemented in connection with any method, kit, reagent, or composition of the invention, and vice versa. Additionally, the compositions of the invention can be used to achieve the methods of the invention.

It should be understood that the specific embodiments described herein are presented by way of illustration and not as limitations of the invention. The main features of the invention can be utilized in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. there is

In the claims and/or specification, the use of the word "a" or "an" in conjunction with the term "comprising" can mean "a" but also Also consistent with the meanings of "one or more," "at least one," and "one or more than one." The use of the term "or" in a claim is used to mean "and/or" unless explicitly indicated to refer only to alternatives or alternatives are not mutually exclusive. provided, however, this disclosure supports definitions that refer only to alternatives and to "and/or." Throughout this application, the term "about" is used to imply that a value includes the inherent error variability of the device, the method utilized to determine the value, or the variability present in the subject of study. ing.

As used herein and in the claims, the word "comprising" (and any form of including, e.g., "comprises" and "comprises"), "having "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes") and "include") or "containing" (and any form of containing, e.g., "contains" and "contain") are defined as inclusive or open end and do not exclude additional, unrecited features, elements, components, groups, integers and/or steps, other unrecited features, elements, components, groups, integers and/or The existence of processes is not excluded. In any of the embodiments of the compositions and methods provided herein, "comprising" means "consisting essentially of" or "consisting of" can be replaced. As used herein, the term "consisting" refers to a recited integer (e.g., feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g., feature ( ), element(s), feature(s), property(s), method/process steps or limitation(s)) are used solely to indicate the presence. As used herein, the phrase "consisting essentially of" requires the specified features, elements, components, groups, integers, and/or steps, but not other the presence of undescribed features, elements, constituents, radicals, integers and/or steps of and those that do not materially affect the basic and novel feature(s) and/or function of the claimed invention do not exclude.

The term "or combinations thereof," as used herein, refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" means at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in the particular context, BA, CA , CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, eg, BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. Those of ordinary skill in the art understand that there is generally no limit to the number of items or terms in any combination unless otherwise apparent from the context.

As used herein, words of approximation, such as, without limitation, “about,” “substantially,” or “substantially,” are not necessarily absolute or Refers to a state that is considered to be a less than perfect state, but one skilled in the art believes that the state is almost fully justified to describe it as existing. The extent to which the description can vary depends on how large a change can be introduced while still allowing one skilled in the art to recognize that the modified feature still possesses the required characteristics and capabilities of the unmodified feature. Generally, but subject to the preceding discussion, numerical values herein modified by a word of approximation, e.g. , may vary by 2, 3, 4, 5, 6, 7, 10, 12 or 15%, or is within normal tolerances in the art, e.g., within 2 standard deviations of the mean It is believed that. All numerical values provided herein are modified by the term about, unless it is clear from the context.

Additionally, section headings herein are provided to be consistent with the suggestions by 37 CFR 1.77 or otherwise to provide organizational clues. These headings shall not limit or characterize the invention(s) set forth in any claims that may issue from this disclosure. Specifically and by way of example, although the heading refers to the "field of the invention," such claims should be limited by the language under this heading that describes the so-called technical field. isn't it. Furthermore, the description of a technology in the "Background of the Invention" section should not be construed as an admission that that technology is prior art to any invention(s) in this disclosure. Neither should the "Summary of the Invention" be considered a characterization of the invention(s) set forth in the issued claims. Furthermore, any reference to "invention" in this disclosure, even in the singular, should not be used to assert that there is only a single point of novelty in this disclosure. Inventions may be set forth in accordance with the limitations of the claims issuing from this disclosure, and such claims shall therefore cover the invention(s) protected thereby, and any equivalents thereto. Define things. In all cases, such claims shall be considered to their own merits in light of the present disclosure and should not be constrained by the headings set forth herein.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described as preferred embodiments, variations can be made in the compositions and/or methods and methods described herein without departing from the concept, spirit and scope of the invention. It will be apparent to those skilled in the art that any method step or order of steps may be applied. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

In order to assist the Patent Office and any reader of any patent that may arise from this application to interpret the claims appended hereto, Applicants have designated "means for" or "for Unless the words "the step of" are expressly used in a particular claim, any of the appended claims may be construed as S. C. § 112, paragraph 6, U.S.C. S. C. We wish to note that we do not intend to invoke §112(f), or equivalents as they exist at the filing date thereof.

For each claim, each dependent claim, to any and all claims, so long as the preceding claim provides a proper antecedent basis for the claim term or element: Both the independent claim and each of the previous dependent claims can be dependent.

References
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Claims (42)

A method of treating a proliferative disorder in a subject with mutated or constitutively active FLT3 who is relapsed/refractory to one or more prior tyrosine kinase inhibitors There is
obtaining a tumor sample from said subject that is relapsed/refractory to one or more previous tyrosine kinase inhibitors;
measuring expression of a mutated or constitutively active FLT3 mutant in said tumor sample; and a therapeutically effective amount of crenolanib sufficient to treat said proliferative disorder or a pharmaceutically acceptable A method comprising administering a salt thereof to said subject. FLT3-TKD; activating mutation in FLT3; copy number increase or amplification of FLT3 gene; or gene comprising fusion of FLT3 with another gene 2. The method of claim 1, which is at least one of fusion. Subjects included midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279, FYSYN, NMS- 03592088, or TG02 citrate; or said subject has an FLT3 mutation that confers resistance to said prior tyrosine kinase inhibitor. described method. amino acid residues K429, A627, N676, A680, F691, Y693, G697, D698, N701, D835, N841, Y842, where the resistance-conferring FLT3 mutation is present alone or in combination with the FLT3-ITD mutation; 4. The method of claim 3, selected from missense mutations that occur in at least one of A848. The resistance-conferring FLT3 mutation was present prior to administration of a previous tyrosine kinase inhibitor, or said resistance-conferring FLT3 mutation was acquired during or after administration of said previous tyrosine kinase inhibitor. 4. The method of claim 3. Proliferative disorders include gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, salivary gland cancer , small cell lung cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancies. A therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is about 50-500 mg per day, 100-450 mg per day, 200-400 mg per day, 300-500 mg per day, 350-500 mg per day, or The method of claim 1, wherein the amount is 400-500 mg. 2. The method of claim 1, wherein the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered at least one of continuously, intermittently, systemically, or locally. 2. The method of claim 1, wherein a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered orally, intravenously, or intraperitoneally. 2. The method of claim 1, wherein the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered up to three times daily or more for as long as the subject is in need of treatment for the proliferative disorder. . A therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, comprising
provided at least one of sequentially or concurrently with another agent to maintain existing patient remission;
Provided as a single agent or in combination with another agent in patients with relapsed/refractory proliferative disorders to maintain remission in patients with relapsed/refractory proliferative disorders; provided as a single agent or in combination with another agent to maintain remission in pediatric patients;
The method of claim 1. Clenoranib or a pharmaceutically acceptable salt thereof is Clenoranib besilate, Clenoranib phosphate, Clenoranib lactate, Clenoranib hydrochloride, Clenoranib citrate, Clenoranib acetate, Clenoranib toluenesulfonate, or clenolanib succinate. A method of inhibiting or reducing mutant FLT3 tyrosine kinase activity or expression in a subject suffering from a proliferative disorder, comprising:
identifying subjects who have discontinued prior tyrosine kinase inhibitor therapy due to refractory or recurrent proliferative disease;
obtaining a tumor sample from said subject;
measuring the expression of mutated FLT3 or a constitutively active FLT3 mutant in said tumor sample; and if said subject has said mutated FLT3 or constitutively active FLT3 mutant. administering to said subject a therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof, wherein said crenolanib or salt thereof reduces proliferative disorder burden or prevents progression of a proliferative disease. ,Method. FLT3-TKD; activating mutation in FLT3; copy number increase or amplification of FLT3 gene; or gene comprising fusion of FLT3 with another gene 14. The method of claim 13, which is at least one of fusion. Subjects included midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279, FYSYN, NMS- 03592088, or a previous tyrosine kinase inhibitor selected from TG02 citrate; or said subject has a FLT3 mutation that confers resistance to said previous tyrosine kinase inhibitor. described method. Amino acid residues K429, A627, N676, A680, F691, where the subject is relapsed or refractory to previous tyrosine inhibitors and said subject is present alone or in conjunction with FLT3-ITD mutations; 14. The method of claim 13, having a resistance-conferring FLT3 mutation selected from missense mutations occurring in at least one of Y693, G697, D698, N701, D835, N841, Y842, A848. The resistance-conferring FLT3 mutation was present prior to administration of a previous tyrosine kinase inhibitor, or said resistance-conferring FLT3 mutation was acquired during or after administration of said previous tyrosine kinase inhibitor. 17. The method of claim 16. Proliferative disorders include gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, salivary gland cancer , small cell lung cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancies. A therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is about 50-500 mg per day, 100-450 mg per day, 200-400 mg per day, 300-500 mg per day, 350-500 mg per day, or 14. The method of claim 13, which is 400-500 mg. 14. The method of claim 13, wherein the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered at least one of continuously, intermittently, systemically, or locally. 14. The method of claim 13, wherein the therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or intraperitoneally. 14. The method of claim 13, wherein the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered up to three times daily or more for as long as the subject is in need of treatment for the proliferative disorder. . A therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, comprising
provided at least one of sequentially or concurrently with another agent to maintain existing patient remission;
Provided as a single agent or in combination with another agent in patients with relapsed/refractory proliferative disorders to maintain remission in patients with relapsed/refractory proliferative disorders; provided as a single agent or in combination with another agent to maintain remission in pediatric patients;
14. The method of claim 13. Clenoranib or a pharmaceutically acceptable salt thereof is Clenoranib besylate, Clenoranib phosphate, Clenoranib lactate, Clenoranib hydrochloride, Clenoranib citrate, Clenoranib acetate, Clenoranib toluenesulfonate, or clenolanib succinate. A method for treating a subject suffering from a proliferative disorder comprising:
To determine whether the patient has a genetic mutation in the FLT3 gene, an alteration in the kinase activity of the FLT3 tyrosine kinase, an overexpression of the FLT3 tyrosine kinase, or an alteration in the FLT3 tyrosine kinase phenotype or genotype;
by obtaining or obtaining a biological sample from said patient; and by performing or performing an assay on said biological sample;
determining whether the subject has increased FLT3 tyrosine kinase activity;
treating said patient with a first tyrosine kinase inhibitor; and if said patient is refractory or relapses after said first tyrosine kinase inhibitor and said patient has a genetic mutation in FLT3; overexpression of FLT3, or alteration in the genotypic phenotype of FLT3 tyrosine kinase, discontinue administration of said first tyrosine kinase inhibitor and administer an effective amount of crenolanib to said patient in vivo. administering to reduce proliferative disorder burden or prevent progression of a proliferative disease. FLT3-TKD; activating mutation in FLT3; copy number increase or amplification of FLT3 gene; or gene comprising fusion of FLT3 with another gene 26. The method of claim 25, which is at least one of fusion. Subjects included midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279, FYSYN, NMS- 26. The subject has been provided with a previous tyrosine kinase inhibitor selected from 03592088, or TG02 citrate; or said subject has a FLT3 mutation that confers resistance to a previous tyrosine kinase inhibitor. the method of. Amino acid residues K429, A627, N676, A680, F691, Y693, G697, D698, where a mutation in the FLT3 gene or an alteration in the FLT3 phenotype or genotype is present alone or in combination with the FLT3-ITD mutation 26. The method of claim 25, wherein the mutation is a resistance-conferring mutation selected from missense mutations that occur in at least one of N701, D835, N841, Y842, A848. The resistance-conferring FLT3 mutation was present prior to administration of a previous tyrosine kinase inhibitor, or said resistance-conferring FLT3 mutation was acquired during or after administration of said previous tyrosine kinase inhibitor. 29. The method of claim 28. Proliferative disorders include gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, salivary gland cancer , small cell lung cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancies. A therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is about 50-500 mg per day, 100-450 mg per day, 200-400 mg per day, 300-500 mg per day, 350-500 mg per day, or 26. The method of claim 25, which is 400-500 mg. 26. The method of claim 25, wherein the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered at least one of continuously, intermittently, systemically, or topically. 26. The method of claim 25, wherein the therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof is administered orally, intravenously or intraperitoneally. 26. The method of claim 25, wherein the therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, is administered up to three times daily or more for as long as the subject is in need of treatment for the proliferative disorder. . A therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, comprising
provided at least one of sequentially or concurrently with another agent to maintain existing patient remission;
Provided as a single agent or in combination with another agent in patients with relapsed/refractory proliferative disorders to maintain remission in patients with relapsed/refractory proliferative disorders; provided as a single agent or in combination with another agent to maintain remission in pediatric patients;
26. The method of claim 25. Crenolanib or a pharmaceutically acceptable salt thereof is crenolanib besilate, crenolanib phosphate, crenolanib lactate, crenolanib hydrochloride, crenolanib citrate, crenolanib acetate, crenolanib toluenesulfonate, or 26. The method of claim 25, which is klenoranib succinate. A method for treating a subject with leukemia comprising:
obtaining a sample from said subject;
determining from said subject sample that the patient has a dysregulated FLT3 receptor or a constitutively active FLT3 receptor;
further determining that said subject is refractory to administration or has relapsed after administration of a previous tyrosine kinase inhibitor; and treating said subject in need of such treatment for leukemia. administering a therapeutically effective amount of crenolanib, or a pharmaceutically acceptable salt thereof, sufficient for
A method, including FLT3-TKD; activating mutation in FLT3; copy number increase or amplification of FLT3 gene; or gene comprising fusion of FLT3 with another gene 38. The method of claim 37, which is at least one of fusion. Subjects included midostaurin, sorafenib, gilteritinib, quizartinib, pexidartinib, FF-10101, CG-806, restortinib, AG1295, AG1296, CEP-5214, CEP-7055, HM43239, pacritinib, MAX-40279, FYSYN, NMS- 38. The subject has been provided with a previous tyrosine kinase inhibitor selected from 03592088, or TG02 citrate; or said subject has a FLT3 mutation that confers resistance to a previous tyrosine kinase inhibitor. the method of. Amino acid residues K429, A627, N676, A680, F691, Y693, G697, D698, where a mutation in the FLT3 gene or an alteration in the FLT3 phenotype or genotype is present alone or in combination with the FLT3-ITD mutation 38. The method of claim 37, wherein the mutation is a resistance-conferring mutation selected from missense mutations that occur in at least one of N701, D835, N841, Y842, A848. The resistance-conferring FLT3 mutation was present prior to administration of a previous tyrosine kinase inhibitor, or said resistance-conferring FLT3 mutation was acquired during or after administration of said previous tyrosine kinase inhibitor. 38. The method of claim 37. Leukemia includes Hodgkin's disease, myeloma, acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic neutrophilic leukemia (CNL); acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL), prolymphocytic leukemia (PML); juvenile myelomonocytic leukemia (JMML); adult T-cell ALL, acute myeloid leukemia (AML), tricytic marrow 38. The method of claim 37, selected from at least one of AML with dysplasia, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), or multiple myeloma (MM).